The primary features of sickle cell disease are events resulting in low flow, poor perfusion and blood and tissue hypoxia that are both pre-disposing to and the consequence of vaso-occlusion. We will test the overall hypothesis that BH4 (tetra-hydrobiopterin, an essential co-factor of nitric oxide synthase (NOS)) levels are decreased in sickle cell disease and in sickle transgenic mice and treatment protective of tissue BH4 levels can have greater efficacy than arginine supplementation alone to interrupt the cycle of polymerization, vaso-occlusion and inflammation that is the origin of pathology in sickle cell disease. Depletion of either arginine or BH4 results in loss of NOS NO generating capacity and production shifts to superoxide, resulting in a vicious cycle in which BH4 is oxidized and more superoxide is generated. We have demonstrated that BH4 is depleted in sickle transgenic mice due in part to oxidative stress. NOS is up regulated by hypoxia via a HIF-sensitive, hypoxia-response element. We further speculate that failure to simultaneously up regulate BH4 will increase oxidative stress. Phosphorylation at serine 1177 and threonine 495 modulate NOS activity. We have demonstrated that the percent phosphorylation at serine 1177 is reduced in mild sickle transgenic mice when they are exposed to hypoxia. We speculate that simultaneous reduction of BH4 and serine phosphorylation will severely impact NOS activity. Hemolysis is thought to be a major factor in reduction of NO bioavailability, both through reaction of cell free hemoglobin with NO and through release of argininase that depletes arginine, the substrate of NOS. We have demonstrated that arginine supplementation reduces hemolysis and oxidative stress in sickle transgenic mice, in part, via inhibition of the Gardos channel. Inhibition of the Gardos channel prevents red cell dehydration and inhibits polymer formation and hemolysis that further reduces NO bioavailability. However, arginine supplementation does not completely correct the vasculopathy observed in sickle transgenic mice. We hypothesize that some of the benefit of arginine supplementation may be due to preservation of BH4. To further test this hypothesis, we are breeding mice that over-express GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in BH4 synthesis into our sickle transgenic strains. These observations may be applicable to patient interventions.